Permeability‐Engineered Compartmentalization Enables In Vitro Reconstitution of Sustained Synthetic Biology Systems

Abstract In nature, biological compartments such as cells rely on dynamically controlled permeability for matter exchange and complex cellular activities. Likewise, the ability to engineer compartment permeability is crucial for in vitro systems to gain sustainability, robustness, and complexity. However, rendering in vitro compartments such a capability is challenging. Here, a facile strategy is presented to build permeability‐configurable compartments, and marked advantages of such compartmentalization are shown in reconstituting sustained synthetic biology systems in vitro. Through microfluidics, the strategy produces micrometer‐sized layered microgels whose shell layer serves as a sieving structure for biomolecules and particles. In this configuration, the transport of DNAs, proteins, and bacteriophages across the compartments can be controlled an guided by a physical model. Through permeability engineering, a compartmentalized cell‐free protein synthesis system sustains multicycle protein production; ≈100 000 compartments are repeatedly used in a five‐cycle synthesis, featuring a yield of 2.2 mg mL−1. Further, the engineered bacteria‐enclosing compartments possess near‐perfect phage resistance and enhanced environmental fitness. In a complex river silt environment, compartmentalized whole‐cell biosensors show maintained activity throughout the 32 h pollutant monitoring. It is anticipated that permeability‐engineered compartmentalization should pave the way for practical synthetic biology applications such as green bioproduction, environmental sensing, and bacteria‐based therapeutics.


Strains and chemicals
Chemicals used in this study were purchased from Sigma Aldrich unless otherwise stated. The bacteria strains used in this study are listed in Supplementary Table 1.

Microfluidic device fabrication
The microfluidic devices (supplementary Fig. 1) were designed using AutoCAD. The layout was printed as dark-field plastic photomasks. The devices were fabricated through polydimethylsiloxane (PDMS)-based soft lithography. First, SU-8 3025 photoresist (Microchem) was spin-coated on a 3-inch silicon wafer at a desired thickness (~30 µm) following the manufacturer's instructions. After a pre-bake of 20 min at 95 °C, the wafer was covered with photomasks and exposed under a 120 mW UV lamp (M365L2, Thorlabs) for 125 s. The wafer was post-baked for 5 minutes at 95 °C and then developed in a SU-8 developer solution (MicroChem) for 10 minutes.
The fabricated silicon master was further cleaned with isopropanol and ethanol and blow-dried with a nitrogen gun. PDMS was prepared by mixing its precursor with a curing agent (SYLGARD 184, Dow Corning) at 10:1 (w:w). After vacuum degassing, the PDMS was poured over the master, followed by a curing process of 4 h at 60 °C.
The cured PDMS slabs were peeled off from the master, and the inlet/outlet ports were created using a custom-made 0.7 mm hole puncher. With oxygen plasma treatment, each slab was bonded to a clean glass slide. Finally, the fabricated devices were treated with Aquapel (PPG Industries) to render the channel surfaces hydrophobic before use.

Compartment manufacturing and characterization
Manufacturing of the compartments started with generating the core hydrogel beads.  Fig. 2a), the core gel beads, pre-solution of the shell layer, and HFE-7500 carrying oil were pumped into a dual-junction flow-focusing device (supplementary Fig. 1b) at typical flow rates of 100, 300 and 700 µL/h, respectively.
The flowrate of shell pre-solution was adjusted between 50 to 300 µL/h to tune the shell thickness. Gelling and de-emulsification steps were then performed as described above. The composition, gelling and re-dissolving conditions for the hydrogels are detailed in Supplementary Table 2.
The re-dissolving of compartment cores was first tested on single-layer PAAm-N,N'bis(acryloyl)cystamine (BAC) gel beads ( Supplementary Fig. 3). BAC is a reversible crosslinker because it carries a disulfide bond which can be broken by DTT. As confirmed under a bright field, the beads were successfully dissolved in 90 s ( Supplementary Fig. 3a). The dissolving process was also confirmed with fluorescent microscopy ( Supplementary Fig. 3b). To render the bead fluorescently labeled,   To confirm the formation of the cavity in the layered microgels, the Cy3-DNA labeled single-layer beads were further manufactured as two-layered core-shell architecture   Supplementary Fig. 6). The cleaning involved three rounds of buffer replacement and incubation. In each round, the sample tubes were placed on a custom-made magnet rack to pull down the beads. The supernatant was then replaced with 1X Tris-HCl buffer, followed by a 5-min incubation. Gel electrophoresis (1% TAE agarose gel) was performed on the compartments to determine that desired fragments were amplified ( Supplementary Fig. 8). The samples were finally examined under the FAM channel of a digital droplet PCR reader (Nebula, Zhejiang ThunderBio Innovation Ltd.) to extract the fluorescence of individual compartments (Supplementary Fig. 9).
The primer pairs targeting 150-1187 bp fragments on the plasmid were designed using an online tool PRIMER3. All the primers were purchased from Genewiz Co. Ltd.
Their sequences are listed in Supplementary
For multicycle protein synthesis, the magnetic compartments were purified from a 45 µL compartmentalized CFPS assay on a custom-made magnet rack after 6-hour incubation and immediately immersed in a new tube containing the CFPS mixture.
The supernatant of each reaction was collected to analyze sfGFP yield. To quantify the absolute mass concentration of sfGFP, a standard curve describing the sfGFP mass concentration versus the fluorescence intensity was derived ( Supplementary Fig.   12). To obtain the curve, His-tagged sfGFP expressed in vivo was purified by a nickel column and then diluted into 45 µL CFPS mixture (excluding the sfGFP plasmid) at varying concentrations. The fluorescence of known sfGFP concentrations was read by a plate reader (BioTek synergy H1). Supplementary Fig. 13a shows the sfGFP yield along a 6-hour CFPS reaction with only cell extract in the compartment cavity and with extra 2.5 U of purified T7 RNA polymerase. Supplementary Fig. 13b compares the yiled of a PeCS-based CFPS system and a bulk system. The sequence structure of the sfGFP plasmid used in this study is illustrated in Supplementary Fig. 14.

Compartmentalized living bacteria-based biosensors
To constructed the compartmentalized living bacteria-based biosensors, the core-shell architecture compartments were used. The compartments (50 µm core diameter, 10 µm shell thickness) were composed by 1% ultra-low agarose gel as core and 2% ultra- Where M is the number of monomers of a polymer molecule and b is monomer size.
In our model, we approximate that a DNA molecule cannot transport across the hydrogel shell when its diameter (double of its R G ) is greater than the hydrogel critical pore size (D p ). The confined-to-diffusible transition takes place in a critical situation where (S3) Combining Eqs. S1-S3, we can derive the relation between a given D p and the critical number of monomers of a DNA molecule right experiencing the confined-todiffusible transition (M crit ), given as Eq. S4 divides the diffusible regime and confined regime of DNA transport through the compartment shell. For DNA, a typical estimation for b K and b is that b K ≈ 100 nm and b ≈ 0.34 nm 7 .

b) protein transport model
In the protein transport model, we consider the gyration radius of a globular protein (R Gp ) as 8

(S5)
Where N denotes the total number of amino acids of a protein molecule. Eq. S5 performed an excellent estimation of a dataset of ~1000 globular proteins from the RCSB protein data bank database (https://www.rcsb.org/). Similar to the DNA model, we can derive the relation between a given D p and the critical number of amino acids of a protein molecule right experiencing the confined-to-diffusible transition (N crit ), given as